The Cu,Zn-superoxide dismutase (SOD1) exerts neuroprotective effects in Amyotrophic Lateral Sclerosis through the Ca2+/ERK1/2/Akt prosurvival pathway

Amyotrophic lateral sclerosis (ALS) is a human adult-onset neurodegenerative disease characterized by progressive weakness, muscles atrophy, spasticity and paralysis resulting in respiratory arrest. These symptoms are related to degeneration and loss of upper motor neurons in cerebral cortex and lower motor neurons in brainstem and spinal cord. Most of cases of ALS are sporadic (sALS), while in the 5-10% of cases the disease is familiar (fALS) with autosomic dominant inheritance. In particular, in more than 15% of cases, the fALS is caused by mutations in the gene coding for the Cu,Zn-superoxide dismutase SOD1. Today, more than 125 different mutations in this gene are known, but how SOD1 caused motor neurons degeneration is unclear. Interestingly, most of mutants partially preserve their enzymatic activity, suggesting that SOD1 mutations are associated with a gain of toxic function rather than a loss of function. For instance, G93A mutation, that has been studied very intensely, leaves the enzyme activity intact. At last, while wt SOD1 secretion is compromise in ALS, mutant SOD1 is not secreted and accumulates in motor neurons within the endoplasmic reticulum and Golgi apparatus. Furthermore, after secretion, SOD1, independently from its dismutase activity, induces phospholipase C (PLC)/ protein kinase C (PKC) transductional pathway. Very interestingly, SOD1 is released by microglial cells and protect against 6-hydroxy-dopamine (6OHDA) toxin. Accordingly, wt SOD1 administration in transgenic SOD1G93A mice ameliorates motor symptoms of disease with an unknown mechanism. Thus, starting from the hypothesis that the neuroprotective effects of SOD1 could be due to its transductional property, we studied the effects of SOD1 and ApoSOD, which lacks dismutase activity, in both NSC-34 motor neurons and primary motor neurons exposed to the cicad neurotoxin L-beta-methylaminoalanine (L-BMAA), a model to reproduce ALS in vitro, and in NSC-34 motor neurons exposed to chemical hypoxia, a model of Ca2+ and ROS-dependent neurodegeneration. We showed that the exogenous administration of both SOD1 and ApoSOD, but not of human recombinant SOD1G93A, prevented cell death induced by the exposure to 300 µM L-BMAA for 48 hrs or to 45 min chemical hypoxia. On the other hand, MnTMPyP pentachloride, a SOD mimetic drug, failed to protect neurons, thus suggesting that SOD1 exerts neuroprotection independently from its most common enzymatic function. Interestingly both the pharmacological inhibition and the knocking down of MEK by PD98059 and siMEK, respectively, counteracted SOD1-induced neuroprotection. Analogously, the well known inhibitor of PI3'K LY294002 and the dominant negative form of Akt prevented SOD1-induced neuroprotective effect on neuronal death induced by L-BMAA and chemical hypoxia. Accordingly, wt SOD1 and ApoSOD elicited a phosphorylation of ERK1/2 and Akt in motor neurons through an early increase of intracellular Ca2+ concentration, thus suggesting that a Ca2+-dependent activation of ERK1/2 and Akt is mainly involved in SOD1-induced neuroprotective effects. Finally, SOD1 prevents ER stress induced by both L-BMAA and chemical hypoxia, as observed by a reduction of GRP78, CHOP, caspase-12 and caspase-3 levels in motor neurons pre-incubated with SOD1 and then exposed to toxic stimuli. Collectively, our data suggest that SOD1 is able to activate Ca2+/ERK1/2/Akt pro-survival pathway in ALS thus exerting a neuroprotective effects independently from its enzymatic function